Audrey Solgadi

Estimation of illicit drugs consumption by wastewater analysis in Paris area (France)

Illicit drugs consumption is actually an important public health concern that needs to be well defined to be managed. A new method, expressed as sewage epidemiology has been proposed by Daughton and developed by Zuccato. This method involves estimating the consumption from the measurement of drug residues in sewage. Several studies have been carried out, leading to an assessment of drugs consumption in some European countries. This work, carried out in Paris area (France) brings new data to this assessment and allows a comparison of cocaine and MDMA consumptions with European estimations. Four wastewater treatment plants (WWTPs) have been retained for the study, taking into account biological treatment, volume capacity, geographic location and social environment. Cocaine and its major metabolite benzoylecgonine (BZE), amphetamine, 3,4-methylenedioxymethamphetamine (MDMA) and buprenorphine were measured in raw water and WWTP effluent using HPLC-MS/MS after SPE extraction. Amphetamine was rarely detected. Cocaine and BZE were quantified at levels from 5 to 282 ng L(-1) and 15 to 849 ng L(-1), respectively. MDMA and buprenorphine concentrations remained under 20 ng L(-1). Cocaine consumption was estimated from cocaine or BZE concentrations measured in raw water and the results showed significant difference in drug taking during week or weekend. The estimated doses observed in this study are lower than those reported for others countries, especially Spain and Italy. MDMA consumption was estimated at lower levels than cocaine.

A comprehensive study of apixaban's degradation pathways under stress conditions using liquid chromatography coupled to multistage mass spectrometry

Apixaban is a novel anticoagulant drug acting as a direct, selective and reversible inhibitor of the coagulation factor Xa. Forced degradation under stress conditions were carried out in order to establish its stability profile. The drug was shown to be stable under photolytic, thermolytic and oxidative conditions, while under hydrolytic conditions, up to seven degradation products were generated for about 15% of drug degradation. The degradation products have been detected by linear gradient reversed phase high-performance liquid chromatography coupled with a photo diode array and with electrospray ionization tandem mass spectrometry. A combination of multistage mass spectrometry and high-resolution mass spectrometry (HR-MS) allowed the structural elucidation. The product ions of the degradation products were compared to those of the apixaban protonated ion so as to assign the most structures possible. This required a study in depth of the drugs fragmentation pattern, which has not been reported so far. In view of the products formed, it appears that hydrolysis of the oxopiperidine moiety of apixaban occurred in acidic medium, whereas that of the tetrahydro-oxo-pyridine moiety would further happen under alkaline conditions. Asides from characterization, the LC method was shown to indicate stability and validated as per the criteria described by the ICH guidelines.

Cardiolipin plays an essential role in the formation of intracellular membranes in Escherichia coli

Mitochondria, chloroplasts and photosynthetic bacteria are characterized by the presence of complex and intricate membrane systems. In contrast, non-photosynthetic bacteria lack membrane structures within their cytoplasm. However, large scale over-production of some membrane proteins, such as the fumarate reductase, the mannitol permease MtlA, the glycerol acyl transferase PlsB, the chemotaxis receptor Tsr or the ATP synthase subunit b, can induce the proliferation of intra cellular membranes (ICMs) in the cytoplasm of Escherichia coli. These ICMs are particularly rich in cardiolipin (CL). Here, we have studied the effect of CL in the generation of these membranous structures. We have deleted the three genes (clsA, clsB and clsC) responsible of CL biosynthesis in E. coli and analysed the effect of these mutations by fluorescent and electron microscopy and by lipid mass spectrometry. We have found that CL is essential in the formation of non-lamellar structures in the cytoplasm of E. coli cells. These results could help to understand the structuration of membranes in E. coli and other membrane organelles, such as mitochondria and ER.

Artificial plasma membrane models based on lipidomic profiling.

Phospholipid monolayers are often described as membrane models for analyzing drug-lipid interactions. In many works, a single phosphatidylcholine is chosen, sometimes with one or two additional components. Drug penetration is studied at 30mN/m, a surface pressure considered as corresponding to the pressure in bilayers, independently of the density of lipid molecular packing. In this work, we have extracted, identified, and quantified the major lipids constituting the lipidome of plasma and mitochondrial membranes of retinoblastoma (Y79) and retinal pigment epithelium cells (ARPE-19), using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The results obtained from this lipidomic analysis were used in an attempt to build an artificial lipid monolayer with a composition mimicking that of the plasma membrane of Y79 cells, better than a single phospholipid. The variety and number of lipid classes and species in cell extracts monolayers exceeding by far those of the phospholipids chosen to mimic them, the π-A isotherms of model monolayers differed from those of lipid extracts in shape and apparent packing density. We propose a model monolayer based on the most abundant species identified in the extracts, with a surface compressional modulus at 30mN/m close to the one of the lipid extracts.

Identification of dabigatran etexilate major degradation pathways by liquid chromatography coupled to multi stage high-resolution mass spectrometry

Dabigatran etexilate (DABET) is an oral direct thrombin inhibitor that has been approved for the prevention of blood clot formation. As the active pharmaceutical ingredient (API) may undergo degradation, leading to drug activity loss or to the occurrence of adverse effects associated with degradation products, thorough knowledge of the APIs stability profile is required. Since very few studies have been reported on the drug stability profile, a study related to DABETs behaviour under stress conditions was carried out in order to identify its major degradation pathways. DABET was subjected to hydrolytic (acidic and alkaline), oxidative, photolytic and thermal stress, as per ICH-specified conditions. Up to ten degradation products along with dabigatran, the active metabolite of DABET, were formed and detected by reverse phase liquid chromatography in gradient mode (LC) coupled to UV and mass spectrometry (UV-MS). Structures were determined by elemental composition determination and study of the fragmentation patterns, using high-resolution mass spectrometry in multistage mode (HR-MSn). Under hydrolytic stress conditions, O-dealkylation may occur and formation of benzimidic acid derivatives was also observed. DABET was shown to be much less susceptible to photolysis and oxidative stress, even if N-dealkylation was highlighted. In view of the structures identified, various degradation pathways of DABET have been proposed.

Supramolecular Chitosan Micro-Platelets Synergistically Enhance Anti-Candida albicans Activity of Amphotericin B Using an Immunocompetent Murine Model

PurposeThe aim of this work is to design new chitosan conjugates able to self-organize in aqueous solution in the form of micrometer-size platelets. When mixed with amphotericin B deoxycholate (AmB-DOC), micro-platelets act as a drug booster allowing further improvement in AmB-DOC anti-Candida albicans activity.MethodsMicro-platelets were obtained by mixing oleoyl chitosan and α-cyclodextrin in water. The formulation is specifically-engineered for mucosal application by dispersing chitosan micro-platelets into thermosensitive pluronic® F127 20 wt% hydrogel.ResultsThe formulation completely cured C. albicans vaginal infection in mice and had a superior activity in comparison with AmB-DOC without addition of chitosan micro-platelets. In vitro studies showed that the platelets significantly enhance AmB-DOC antifungal activity since the IC50 and the MIC90 decrease 4.5 and 4.8-times. Calculation of fractional inhibitory concentration index (FICI = 0.198) showed that chitosan micro-platelets act in a synergistic way with AmB-DOC against C. albicans. No synergy is found between spherical nanoparticles composed poly(isobutylcyanoacrylate)/chitosan and AmB-DOC.ConclusionThese results demonstrate for the first time the ability of flattened chitosan micro-platelets to have synergistic activity with AmB-DOC against C. albicans candidiasis and highlight the importance of rheological and mucoadhesive behaviors of hydrogels in the efficacy of the treatment.

Phototransformation patterns of the antiplatelet drug tirofiban in aqueous solution, relevant to drug delivery and storage

Tirofiban is a synthetic, nonpeptidic fibrinogen receptor antagonist used as an antiplatelet drug for intravenous delivery. As the active pharmaceutical ingredient may undergo light exposure during manufacturing, storage and/or delivery, there is a need to acquire an extensive knowledge of its major photochemical-degradation pathways. Thus, photochemical-degradation of tirofiban under simulated light irradiation in aqueous solution and in the absence of photosensitizers or photocatalysts, has been investigated in terms of mechanisms. The structural characterization of the photochemical products was carried out with using performance liquid chromatography-multistage high-resolution mass spectrometry along with on-line hydrogen/deuterium exchange. The identification of the twelve detected photochemical products suggested that the photo-transformation of tirofiban occurred via multiple reaction pathways, initiated either by electron or hydrogen atom transfer. These included the photo-oxidation of the piperidine moiety without impacting the secondary amine, the hydroxylation of the methylene group activated by the aromatic ring, the oxidation of the alkyl-sulfonamide group and also the decarboxylative oxidation of the molecule. Hydroxylated compounds, geminal and vicinal-diol compounds, were highlighted suggesting that most of the photoproducts are more hydrophilic than the drug. Understanding the main photo-degradation routes is a good basis to work out efficient measures so as to mitigate or avoid tirofiban instability.

Rapid analysis of glutamate, glutamine and GABA in mice frontal cortex microdialysis samples using HPLC coupled to electrospray tandem mass spectrometry

&NA; In vivo measurement of multiple neurotransmitters is highly interesting but remains challenging in the field of neuroscience. GABA and L‐glutamic acid are the major inhibitory and excitatory neurotransmitters, respectively, in the central nervous system, and their changes are related to a variety of diseases such as anxiety and major depressive disorder. This study described a simple method allowing the simultaneous LC–MS/MS quantification of L‐glutamic acid, glutamine and GABA. Analytes were acquired from samples of the prefrontal cortex by microdialysis technique in freely moving mice. The chromatographic separation was performed by hydrophilic interaction liquid chromatography (HILIC) with a core‐shell ammonium‐sulfonic acid modified silica column using a gradient elution with mobile phases consisting of a 25 mM pH 3.5 ammonium formate buffer and acetonitrile. The detection of L‐glutamic acid, glutamine and GABA, as well as the internal standards [d6]‐GABA and [d5]‐glutamate was performed on a triple quadrupole mass spectrometer in positive electrospray ionization and multiple reaction monitoring mode. The limit of quantification was 0.63 ng/ml for GABA, 1.25 ng/ml for L‐glutamic acid and 3.15 ng/ml for glutamine, and the intra‐day and inter‐day accuracy and precision have been assessed for the three analytes. Therefore, the physiological relevance of the method was successfully applied for the determination of basal extracellular levels and potassium‐evoked release of these neuroactive substances in the prefrontal cortex in adult awake C57BL/6 mice.

Pemetrexed degradation by photocatalytic process: Kinetics, identification of transformation products and estimation of toxicity

This study employed a UV-A/visible/TiO2 system to investigate the degradation of pemetrexed, an antifolate agent used in chemotherapy. The laboratory-scale method employed a photostability chamber that could be used to study multiple samples. Reversed-phase HPLC coupled with high-resolution ESI-LTQ-Orbitrap mass spectrometry was used to determine the transformation products (TPs) of PEME. Based on the identified TPs and existing chemical knowledge, the mechanism of degradation of the target compound was proposed. Concentrations were monitored as a function of time, and the degradation kinetics were compared. The structures of seven TPs, four of which have not been described to date, were proposed. Most of the TPs stemmed from OH radical additions to the dihydropyrrole moiety and oxidative decarboxylation of the glutamate residue. Based on the elucidated structures, a computational toxicity assessment was performed, showing that the TPs with higher log D values than the parent compound are more toxic than the PEME itself. To support these findings, the toxicities of irradiated samples on Vibrio fischeri were monitored over time. The experimental results corresponded well with the results of previous computational studies.

Optimization of normal phase chromatographic conditions for lipid analysis and comparison of associated detection techniques

One important challenge in lipid class analysis is to develop a method suitable or, at least adaptable, for a vast diversity of samples. In the current study, an improved normal-phase liquid chromatography (NPLC) method allowed analyzing the lipid classes present in mammalian, vegetable as well as microorganism (yeast and bacteria) lipid samples. The method effectively separated 30 lipid classes or subclasses with a special focus on medium polarity lipids. The separation was carried out with bare silica stationary phase and was coupled to evaporative light scattering detection (ELSD), charged aerosol detection (Corona-CAD®) and mass spectrometry. Solutions are provided to circumvent technical issues (such as pumping solvents of low viscosity, solvent purity, rinsing step). The influence of mobile phase composition and addition of ionic modifiers on the chromatographic behavior of particular lipid classes is documented. A comparison between ELSD and Corona-CAD® confirmed the interest of this later detector for samples with a wide range of concentration of different lipids. Three common atmospheric pressure ionization interfaces were used for coupling the NPLC separation to a LTQ Velos Pro® mass spectrometer. The comparison of the chromatographic profiles showed that atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) are both suitable to detect the different lipid classes whereas APPI allows a better sensitivity for lipids at low-concentration.